P53 Upregulated Modulator Of Apoptosis Protein Expression Research Articles

The effect of Oxaliplatin in leading to apoptosis of intestinal cancer cells byinducing PUMA protein expression through reactive oxygen species

Previous studies demonstrated Oxaliplatin, a drug used in treating intestinal cancers by inducing cancer cell apoptosisregulated by p53 upregulated modulator of apoptosis (PUMA) protein gene expression. Increasing the reactiveoxygen species (ROS) level is possible to speed up the expression of PUMA protein in Oxaliplatin. This study aimsto investigate the effect of Oxaliplatin in intestinal cancer cells, leading to its apoptosis by inducing PUMA proteinexpression through ROS, compared to Cisplatin and PBS. The result of the study will provide important insight into thepreclinical effectiveness of Oxaliplatin in intestinal cancer cells, and it also testifies to the underlying mechanism of thisdrug. Future studies should focus on investigating drug combinations with Oxaliplatin that provides synergism towardthe disease and decreases its toxicity.

MiR-22 protect PC12 from ischemia/reperfusion-induced injury by targeting p53 upregulated modulator of apoptosis (PUMA)

ABSTRACT MicroRNAs have been implicated as critical regulatory molecules in many cerebrovascular diseases. Recent studies demonstrated miR-22 might provide a potential neuroprotective effect. However, the neuroprotective effect of miR-22 in ischemia/reperfusion (I/R) injury has not been thoroughly elucidated. In this study, the PC12 cells were subjected to 4 h oxygen and glucose deprivation (I) and 24 h reoxygenation (R). The PC12 cells were pre-transfected with miR-22 or anti-miR-22 or siRNA-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA)(PUMA siRNA) or their controls at 24 h prior to exposure to I/R. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot were employed to analyze mRNA and protein expression. PI and Annexin V assays and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to quantify the rate of apoptosis. We found that miR-22 expression was significantly downregulated in the PC12 cells subjected to I/R. Loss of function of miR-22 increased PC12 apoptosis after I/R, and overexpression of miR-22 decreases PC12 apoptosis after I/R. PUMA protein was upregulated in the I/R group as compared with the sham group. The increased PUMA protein expression and apoptosis induced by I/R was reversed by transfection with PUMA siRNA. We concluded that I/R enhanced apoptosis and PUMA expression in PC12 cells via downregulation of miR-22. Enhanced miR-22 expression reversed both PUMA expression and apoptosis induced by I/R in PC12 cells. miR-22/PUMA axis has important implications for their clinical applications.

Inhibiting aberrant p53-PUMA feedback loop activation attenuates ischaemia reperfusion-induced neuroapoptosis and neuroinflammation in rats by downregulating caspase 3 and the NF-\u03baB cytokine pathway

BackgroundIschaemia reperfusion (IR) induces multiple pathophysiological changes. In addition to its classical role in regulating tumourigenesis, the feedback loop formed by p53 and its driven target p53-upregulated modulator of apoptosis (PUMA) was recently demonstrated to be the common node tightly controlling various cellular responses during myocardial IR. However, the roles of the p53-PUMA feedback loop in the spinal cord remain unclear. This study aimed to elucidate the roles of p53-PUMA feedback interactions in the spinal cord after IR, specifically investigating their regulation of caspase 3-mediated apoptosis and nuclear factor (NF)-κB-mediated cytokine release.MethodsSD rats subjected to 12 min of aortic arch occlusion served as IR models. Neurological assessment as well as p53 and PUMA mRNA and protein expression analyses were performed at 12-h intervals during a 48-h reperfusion period. The cellular distributions of p53 and PUMA were determined via double immunofluorescence staining. The effects of the p53-PUMA feedback loop on modulating hind-limb function; the number of TUNEL-positive cells; and protein levels of caspase 3, NF-κB and cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, were evaluated by intrathecal treatment with PUMA-specific or scramble siRNA and pifithrin (PFT)-α. Blood-spinal cord barrier (BSCB) breakdown was examined by Evans blue (EB) extravasation and water content analyses.ResultsIR induced significant behavioural deficits as demonstrated by deceased Tarlov scores, which displayed trends opposite those of PUMA and p53 protein and mRNA expression. Upregulated PUMA and p53 fluorescent labels were widely distributed in neurons, astrocytes and microglia. Injecting si-PUMA and PFT-α exerted significant anti-apoptosis effects as shown by the reduced number of TUNEL-positive cells, nuclear abnormalities and cleaved caspase 3 levels at 48 h post-IR. Additionally, p53 colocalized with NF-κB within the cell. Similarly, injecting si-PUMA and PFT-α exerted anti-inflammatory effects as shown by the decreased NF-κB translocation and release of IL-1β and TNF-α. Additionally, injecting si-PUMA and PFT-α preserved the BSCB integrity as determined by decreased EB extravasation and spinal water content. However, injecting si-Con did not induce any of the abovementioned effects.ConclusionsInhibition of aberrant p53-PUMA feedback loop activation by intrathecal treatment with si-PUMA and PFT-α prevented IR-induced neuroapoptosis, inflammatory responses and BSCB breakdown by inactivating caspase 3-mediated apoptosis and NF-κB-mediated cytokine release.

Role of PUMA in methamphetamine-induced neuronal apoptosis.

Exposure to methamphetamine (METH), a widely used illicit drug, has been shown to cause neuron apoptosis. p53 upregulated modulator of apoptosis (PUMA) is a key mediator in neuronal apoptosis. This study aimed to examine the effects of PUMA in METH-induced neuronal apoptosis. We determined PUMA protein expression in PC12 cells and SH-SY5Y cells after METH exposure using western blot. We also observed the effect of METH on neuronal apoptosis after silencing PUMA expression with siRNA using TUNEL staining and flow cytometry. Additionally, to investigate possible mechanisms of METH-induced PUMA-mediated neuronal apoptosis, we measured the protein expression of apoptotic markers, including cleaved caspase-3, cleaved PARP, Bax, B-cell leukemia/lymphoma-2 (Bcl-2) and cytochrome c (cyto c), after METH treatment with or without PUMA knockdown. Results showed that METH exposure induced cell apoptosis, increased PUMA protein levels, activated caspase-3 and PARP, elevated Bax and reduced Bcl-2 expression, as well as increased the release of cyto c from mitochondria to the cytoplasm in both PC12 and SH-SY5Y cells. All these effects were attenuated or reversed after silencing PUMA. A schematic depicting the role of PUMA in METH-induced mitochondrial apoptotic pathway was proposed. Our results suggest that PUMA plays an important role in METH-triggered apoptosis and it may be a potential target for ameliorating neuronal injury and apoptosis caused by METH.

MiR-125b protects against ethanol-induced apoptosis in neural crest cells and mouse embryos by targeting Bak 1 and PUMA

MicroRNAs are a class of small noncoding RNAs that have been implicated in regulation of a broad range of cellular and physiologic processes, including apoptosis. The objective of this study is to elucidate the roles of miR-125b in modulating ethanol-induced apoptosis in neural crest cells (NCCs) and mouse embryos. We found that treatment with ethanol resulted in a significant decrease in miR-125b expression in NCCs and in mouse embryos. We also validated that Bcl-2 antagonist killer 1 (Bak1) and p53-upregulated modulator of apoptosis (PUMA) are the direct targets of miR-125b in NCCs. In addition, over-expression of miR-125b significantly reduced ethanol-induced increase in Bak1 and PUMA protein expression, caspase-3 activation, and apoptosis in NCCs, indicating that miR-125b can modulate ethanol-induced apoptosis by the regulation of Bcl-2 and p53 pathways. Furthermore, microinjection of miR-125b mimic resulted in a significant increase in miR-125b expression and a decrease in the protein expression of Bak1 and PUMA in ethanol-exposed mouse embryos. Up-regulation of miR-125b also significantly reduced ethanol-induced caspase-3 activation and diminished ethanol-induced growth retardation in mouse embryos. This is the first demonstration that miR-125b can prevent ethanol-induced apoptosis and that microinjection of miRNA mimic can prevent ethanol-induced embryotoxicity.

Exogenous p53 upregulated modulator of apoptosis (PUMA) decreases growth of lung cancer A549 cells.

To investigate the influence of exogenous p53 upregulated modulator of apoptosis (PUMA) expression on cell proliferation and apoptosis in human non-small cell lung cancer A549 cells and transplanted tumor cell growth in nude mice. A549 cells were divided into the following groups: control, non- carrier (NC), PUMA (transfected with pCEP4- (HA) 2-PUMA plasmid), DDP (10 μg/mL cisplatin treatment) and PUMA+DDP (transfected with pCEP4-(HA)2-PUMA plasmid and 10 μg/mL cisplatin treatment). The MTT method was used to detect the cell survival rate. Cell apoptosis rates were measured by flow cytometry, and PUMA, Bax and Bcl-2 protein expression levels were measured by Western blotting. Compared to the control group, the PUMA, DDP and PUMA+DDP groups all had significantly decreased A549 cell proliferation (p<0.01), with the largest reduction in the PUMA+DDP group. Conversely, the apoptosis rates of the three groups were significantly increased (P<0.01), and the PUMA and DDP treatments were synergistic. Moreover, Bax protein levels significantly increased (p<0.01), while Bcl-2 protein levels significantly decreased (p<0.01). Finally, both the volume and the weights of transplanted tumors were significantly reduced (p<0.01), and the inhibition ratio of the PUMA+DDP group was significantly higher than in the single DDP or PUMA groups. Exogenous PUMA effectively inhibited lung cancer A549 cell proliferation and transplanted tumor growth by increasing Bax protein levels and reducing Bcl-2 protein levels.

Effects of p53 upregulated modulator of apoptosis expression on apoptosis of artificial condrocytes

Objective To explore the effects of p53 upregulated modulator of apoptosis(PUMA)on apoptosis in the mouse knee joint osteoarthritis cartilage. Methods The osteoarthritis cell model was made by using mouse artificial chondrocytes treated with interleukin(IL)-1β, and small interfering RNA(siRNA) of PUMA was transfected into chondrocytes. The expression of PUMA and the apoptosis were detected by Western blotting and Hoechst staining at baseline and after transfection. Results PUMA protein expression in control group was 6.11 times of blank group, and that in control group was 2.02 and 3.30 times of experimental groups. The apoptosis rate in blank group, control group, and experimental groups A and B was 3.6%,29.6%,9.0% and 13.2% respectively. SiRNA of PUMA had been transfected into chondrocytes successfully. PUMA protein expression was suppressed, and apoptosis of chondrocytes decreased. Conclusion PUMA gene has taken part in the apoptosis of chondrocytes in mice with osteoarthritis. Key words: Osteoarthritis; Artificial chondrocytes; p53 upregulated modulator of apoptosisl; Small interfering RNA; Apoptosis

PUMA gene transfection can enhance the sensitivity of epirubicin-induced apoptosis of MCF-7 breast cancer cells.

We explored whether p53 upregulated modulator of apoptosis (PUMA) gene transfection could enhance the sensitivity of epirubicin-induced apoptosis of MCF-7 breast cancer cells. The liposome-mediated recombinant eukaryotic expression vector PU-MA-pCDNA3 and empty vector plasmid were stably transfected into MCF-7 cells. Epirubicin (0.01-100 μM) was applied to MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells for 72 h. The MTT assay was used to calculate the cell survival rate in each group, and the 50% inhibitory concentration (IC50) was calculated. The IC50 values of epirubicin in MCF-7, MCF-7/PUMA, and MCF-7/pCDNA3 cells were 13 ± 1.4, 1.8 ± 0.2, and 10.7 ± 1.3 μM, respectively. The sensitivity of MCF-7/PUMA cells to epirubicin increased 7.2-fold. Epirubicin induced apoptosis in MCF-7 cells dose-dependently, but MCF-7/PUMA cell-induced apoptosis was more significant compared to controls. Low concentrations of epirubicin (0.1 μM) caused low levels of apoptosis of MCF-7/pCDNA3 (1.15 ± 0.26%) and MCF-7 cells (0.9 ± 0.24%), but significantly induced apoptosis of MCF-7/PUMA cells (6.44 ± 1.46%). High epirubicin concentration (1 μM) induced apoptosis in each group, but the epirubicin MCF-7/PUMA apoptosis rate (35.47 ± 9.36%) was significantly higher than that of MCF-7 (12.6 ± 3.73%) and MCF-7/ pCDNA3 (15.2 ± 5.17%) cells (P < 0 01). Flow cytometry and TUNEL assays for apoptosis detection showed similar results. PUMA protein expression in MCF-7/PUMA cells was significantly higher than that in MCF-7 and MCF-7/pCDNA3 cells by Western blot analysis. There-fore, stable transfection of PUMA can significantly enhance epirubicin-induced apoptosis sensitivity of MCF-7 breast cancer cells.

Abstract 8558: Mechanical Stretch Induces the Apoptosis Regulator Puma in Vascular Smooth Muscle Cells

Aims The expression of PUMA (p53-upregulated modulator of apoptosis), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress. How mechanical cyclic stretch affects the regulation of PUMA in vascular smooth muscle cells (VSMCs) during apoptosis is not fully understood. We aimed to test the hypothesis that cyclical stretch enhances PUMA expression in VSMCs undergoing apoptosis. Methods and results Human VSMCs grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. An in vivo model of aorta-caval shunt in adult rats was used to investigate PUMA expression. Cyclic stretch significantly enhanced PUMA protein and mRNA expression after 18 h of stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125 and interferon-γ (IFN-γ) antibody 30 min before stretch inhibited the induction of PUMA protein. Gel shift assay showed that DNA-binding activity of Interferon Regulatory Factor-1 (IRF-1) increased after stretch. SP600125, JNK siRNA and IFN-γ antibody abolished the binding activity induced by stretch. Stretch increased while PUMA-Mut plasmid, SP600125, and IRF-1 antibody abolished the promoter activity. Cyclic stretch significantly increased the interferon-γsecretion from VSMCs at 1 h and remained elevated for 6 h. Both conditioned media from stretched VSMCs and exogenous administration of IFN-γrecombinant protein to the non-stretched VSMCs increased PUMA protein expression similar to that seen after stretch. Cells in the sub-G1 fraction stained with propidium iodide and caspase 3 activity was elevated after stretch for 18 h and addition of interferon-γ. Addition of PUMA and JNK siRNA abolished the induction of sub-G1 and caspase 3 activity induced by stretch. The increases of TUNEL-positive nuclei in VSMCs induced by stretch were significantly reversed by PUMA and JNK siRNA. An in vivo model of aorta-caval shunt in adult rats also demonstrated the increased PUMA protein expression in the aorta. Conclusion Cyclic stretch enhanced PUMA expression in cultured human VSMCs. The stretch-induced PUMA is mediated by IFN-γ,JNK and IRF-1 pathway. These findings suggest that PUMA plays a role in stretch-induced VSMC apoptosis.

Mechanical stretch induces the apoptosis regulator PUMA in vascular smooth muscle cells

The expression of PUMA (p53-up-regulated modulator of apoptosis), an apoptosis-regulating gene, increases during endoplasmic reticulum stress. The mechanisms by which cyclic stretch influences the regulation of PUMA in vascular smooth muscle cells (VSMCs) during apoptosis remain unclear. We hypothesized that cyclic stretch enhances PUMA expression in VSMCs undergoing apoptosis. Human VSMCs grown on a Flexcell I flexible membrane base were stretched via vacuum to 20% of elongation at a frequency of 1 Hz. An in vivo model of volume overload with aorta-caval shunt and pressure overload with aortic banding in adult rats was used to study PUMA expression. Cyclic stretch markedly enhanced PUMA protein and gene expression after stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125 and interferon-γ (IFN-γ) antibody 30 min before stretch inhibited PUMA expression. Gel shift assay demonstrated that stretch increased the DNA binding activity of interferon regulatory factor-1 (IRF-1). SP600125, JNK small interfering RNA, and IFN-γ antibody attenuated the DNA binding activity induced by stretch. PUMA-Mut plasmid, SP600125, and IRF-1 antibody attenuated the promoter activity. Stretch increased secretion of IFN-γ from VSMCs, and conditioned media from stretched VSMCs increased PUMA protein expression. The in vivo model of aorta-caval shunt and aortic banding also showed increased PUMA protein expression in the aorta. Cyclic mechanical stretch increases PUMA expression in cultured human VSMCs. The PUMA expression induced by stretch is mediated by IFN-γ, JNK, and IRF-1 pathways. These findings suggest that PUMA is an important mediator in VSMC apoptosis induced by stretch.

Tumor-specific adenovirus-mediated PUMA gene transfer using the survivin promoter enhances radiosensitivity of breast cancer cells in vitro and in vivo

Survivin is a member of the inhibitors of apoptosis protein family, is expressed in most of human cancers. Thus, we hypothesized that using a survivin promoter-driven vector system, an apoptosis-inducible gene may be preferentially restricted to survivin-positive tumor cells. PUMA (p53 upregulated modulator of apoptosis) was recently identified a potent proapoptotic molecule. In the present study, our aim is to investigate whether adenovirus-mediated PUMA gene transfer using the survivin promoter could specifically enhance radiosensitivity of breast cancer cells in vitro and in vivo. Firstly, we performed RT-PCR assay to detect survivin mRNA expression in four human breast cancer cell lines and two normal cell lines. Then, we constructed plasmid vectors expressing luciferase or EGFP reporter gene driven by the survivin core promoter and evaluated its transcriptional activity in vitro. Next, a survivin promoter-driven adenoviral system expressing PUMA gene was constructed. Western blotting analysis of PUMA protein expression in MCF-7 and MCF210 cells treated with various adenovirus (empty, driven by CMV promoter or survivin promoter). Followingly, the effects of PUMA overexpression on the in vitro radiosensitivity of breast cancer cells were investigated by clonogenic formation assay. Cellular apoptosis were determined by FCM and TUNEL assays. The expression and activities of cellular caspase-3 were assayed by Western blot and colorimetry. Finally, we investigated the effect of PUMA overexpression on the radiosensitivity of breast carcinoma cells in vivo. The survivin mRNA expression was obviously upregulated in breast cancer cell lines but in normal cell lines. Moreover, the survivin promoter could drive high-level expression of luciferase or EGFP in breast cells but not in normal cells. Using a survivin promoter-driven adenoviral system expressing PUMA gene, we found that PUMA expression was specifically and efficiently induced in MCF-7 cell but not in MCF210 cell. Furthermore, we observed that the novel adenovirus system could significantly enhance radiosensitivity of MCF-7 cell in vitro and in vivo, which might be associated with apoptosis induction by activating cellular caspase-3. Our findings indicated that the survivin promoter-driven adenovirus system expression PUMA gene might be explored as a potential tool for radiosensitization of human breast cancer cells with tumor specificity and high efficacy.